Protein degradation through covalent inhibitor-based PROTACs

Article abstract of DOI:10.1039/c9cc08238g

Tremendous advancements in proteolysis targeting chimera (PROTAC) technology have been made in recent years. However, whether a covalent inhibitor-based PROTAC can be developed remains controversial. Here, we successfully developed chimeric degraders based on covalent inhibitors to degrade BTK and BLK kinases, demonstrating that covalent inhibitor-based PROTACs are viable and useful tools.

Full text of DOI:10.1039/c9cc08238g

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Protein degradation through covalent
inhibitor-based PROTACs†
Cite this:DOI: 10.1039/c9cc08238g
Gang Xue, Jiahui Chen, Lihong Liu, Danli Zhou, Yingying Zuo,
Received 21st October 2019,
Accepted 27th December 2019
Tiancheng Fu and Zhengying Pan
*
DOI: 10.1039/c9cc08238g
rsc.li/chemcomm
5
–7
Tremendous advancements in proteolysis targeting chimera indicated that such molecules degrade proteins in cells.
PROTAC) technology have been made in recent years. However, However, a recent report clearly showed that a covalent PROTAC
(
whether a covalent inhibitor-based PROTAC can be developed could not induce the degradation of its target protein – Bruton’s
8
remains controversial. Here, we successfully developed chimeric tyrosine kinase (BTK). Here we present our efforts to develop
degraders based on covalent inhibitors to degrade BTK and BLK covalent inhibitor-based PROTACs that successfully degrade BTK
kinases, demonstrating that covalent inhibitor-based PROTACs are and B lymphocyte kinase (BLK) in live cells.
9
viable and useful tools.
BTK is a non-receptor cytoplasmic tyrosine kinase and
participates in the B-cell receptor (BCR) signaling pathway,
1
0
Targeted protein degradation has been used in the clinic to which plays a key role in B cell lymphomas. The first BTK
1
1
treat patients by using monovalent small molecule degraders, covalent inhibitor, ibrutinib has been approved by the FDA
1
12–15
such as fulvestrant, lenalidomide and pomalidomide. Proteo- for the treatment of various B cell malignancies. BTK PROTACs
lysis targeting chimera (PROTAC) technology emerged by using require a BTK binding moiety connected via a linker to an E3 ligase
2
bivalent chemical agents in 2001, and a PROTAC molecule is ligand. For the BTK binding moiety, the covalent inhibitors
1
6
composed of a protein binding ligand, an E3 ubiquitin ligase ibrutinib and PLS-123 were selected (Chart 1 and Fig. S1,
ligand and a linker connecting the two ligands. PROTACs ESI†), because of their high affinities and the large structural
induce an E3 ligase to recruit the target protein, resulting differences between their scaffolds. Based on our previous work
1
7
18
in target protein polyubiquitination and degradation via the on converting ibrutinib and PLS-123 into fluorescent BTK
ubiquitin–proteasome system. Degradation of the target protein probes, we selected the carboxyl groups on compound 1 and
by PROTACs is normally considered as an event-driven pharma- compound 2 as the egress point to ligate the linkers (Chart 1).
3
cological effect and acts in a catalytic manner. In recent years, For E3 ligase ligands, pomalidomide and VH032, which recruit
PROTACs have made great progress and have been successfully E3 ubiquitin ligase cereblon (CRBN) and VHL, respectively, were
4
applied to degrade various kinds of proteins, including kinases, employed (Fig. S1, ESI†). Based on the X-ray crystal structures of
1
9
nuclear receptors, BET proteins, and other proteins.
CRBN complexed with pomalidomide (PDB ID: 4CI3) and VHL
2
0
Covalent inhibitors have experienced a resurgence during complexed with VH032 (PDB ID: 4W9H), the linker bound to
the past decade both as chemical tools and as approved pomalidomide was ligated at carbon-4 on the phthalimide ring
medicines. We have been interested in combining these two and the linker bound to VHL was ligated through the terminal
powerful modalities to generate covalent inhibitor-based PROTACs. acetamide group of VH032. Thus compounds 3–10 were
In the literature, the degradation results of covalent PROTACs are designed and synthesized (Schemes S1–S4, ESI†).
mixed. The first PROTAC article described a covalent inhibitor-
2
based degrader but was only tested in an in vitro system.
Additional reports on covalently binding PROTACs have
State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical
Genomics, Engineering Laboratory for Chiral Drug Synthesis, School of Chemical
Biology and Biotechnology Shenzhen Graduate School, Peking University,
Xili University Town, PKU Campus, F-311, Shenzhen, 518055, China.
E-mail: panzy@pkusz.edu.cn
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc08238g
Chart 1 Chemical structures of BTK inhibitors used in this study.
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degradation activity (Fig. 1e). However, PROTACs 6a and 6b with
different lengths of carbon chains effectively degraded BTK.
PROTAC 6b had an improved degradation activity with its DC50
(
the concentration causing 50% reduction in the protein level
relative to the vehicle-treated sample) value likely less than
00 nM and Dmax (the maximum reduction of the protein level
3
2
4
relative to the vehicle-treated sample) value of 75% at 1 mM
Fig. 1f).
Since the degradation ability of PROTACs was also impacted
(
2
5
by the E3 ligase, we further replaced the pomalidomide in
PROTACs 3a and 6b with VH032 to employ another E3 ligase
(VHL) and synthesized PROTACs 7 and 8 (Fig. 2a). Indeed,
compound 7 showed the most significant ability to reduce the
BTK protein level with DC50 = 136 nM and Dmax = 88% (Fig. 2b
and c). By contrast, the degradation activity of PROTAC 8 was
significantly lower than that of PROTAC 6b.
Non-covalent inhibitor-based PROTACs 9 and 10 were
obtained by replacing the acrylamide reactive group in PROTACs
6
b and 7 with a propanamide group. Competition assays with
1
7
PROTACs 6b, 7, 9 and 10 and covalent fluorescent probe 11
(Fig. S1, ESI†) were also performed. As expected, preincubation
of PROTACs 6b and 7 with BTK for 1.5 h greatly decreased the
binding of probe 11 with BTK, while 9 and 10 did not (Fig. S3,
ESI†). In a cellular activity assay, the treatment with PROTAC 7
inhibited BTK’s autophosphorylation of Tyr223 even after 7 was
washed-out with fresh medium (Fig. S4, ESI†). Additionally,
compound 7 displayed an incubation time-dependent inhibition
behaviour in a BTK kinase activity assay (Fig. S5, ESI†). Collectively,
these results indicate that PROTACs 6b and 7 are irreversibly bound
to BTK. Head-to-head comparisons were then performed to
Fig. 1 The degradation of BTK by PROTACs 3–6. (a) Chemical structures
of PROTACs 3 and 4. (b) Degradation of BTK by PROTACs with different
linker lengths and linker compositions in K562 cells for 24 h at the two
indicated concentrations. (c) BTK protein levels in cells. Numbers were
calculated by the BTK/GAPDH ratio with normalization by the DMSO
control as 100. (d) Chemical structures of PROTACs 5 and 6. (e) Degradation compare the degradation activities of covalent PROTACs (6b and 7)
of BTK by PROTACs 5 and 6 in K562 cells for 24 h at the two indicated
concentrations. (f) BTK protein levels in cells. Numbers were calculated
and non-covalent PROTACs (9 and 10) (Fig. 3a). The results showed
that the BTK protein levels in cells treated with PROTAC 7 were
significantly lower than those treated with PROTAC 9, while
by the BTK/GAPDH ratio with normalization by the DMSO control as 100.
The bars in the graphs show the means Æ standard deviations from two
biological replicates.
We first connected the ibrutinib analogue 1 to pomalidomide
2
1
with different linkers (3a–d, 4a–d) (Fig. 1a) and examined their
ability to degrade BTK in cells. After treating K562 cells with
increasing concentrations of compounds for 24 h, we analyzed
cell lysates by Western blot. Decreases in BTK protein levels were
observed with the compounds’ concentrations ranging from
100 nM to 3 mM, whereas ibrutinib itself did not reduce the
BTK protein level (Fig. S2, ESI†). However, the level of BTK
protein could only be reduced by approximately 50%. The
degradation effects of the compounds (3a–d, 4a–d) are shown
in Fig. 1b and c and Fig. S2 (ESI†).
Since the ligand-induced protein–protein interaction
between the E3 ligase and target protein is very important
for the degradation efficiency of PROTACs, we conjugated ligands. (a) Chemical structures of PROTACs 7 and 8. (b) Western blot
Fig. 2 The comparison of BTK degradation by PROTACs with different E3
2
2
results of BTK and GAPDH. K562 cells were treated with PROTACs for
pomalidomide with another covalent inhibitor, PLS-123, whose
1
8 h at the two indicated concentrations. (c) BTK protein levels in cells.
scaffold and its binding mode with BTK were different from those
Numbers were calculated by the BTK/GAPDH ratio with normalization by
the DMSO control as 100. The bars in the graphs show the means Æ
standard deviations from three biological replicates. *P o 0.05, **P o 0.01
23
of ibrutinib. Four analogues with different linkers (Fig. 1d) were
synthesized, and their degradation activities were evaluated.
PROTACs with PEG linkers (5a, 5b) did not show appreciable and ***P o 0.001 are from unpaired t-tests.
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To investigate the mechanism of BTK and BLK degradation
using PROTAC 7, cells were preincubated with the E3 ligase-VHL
inhibitor (VH032), the BTK and BLK inhibitor (ibrutinib), the
proteasome inhibitor (carfilzomib) or the ubiquitin-activating
enzyme inhibitor (MLN7243), then it was found that PROTAC 7
could not induce the degradation of BTK (Fig. S8a, ESI†) and
BLK (Fig. S8b, ESI†), which indicates that the degradation
requires the proteasome system and PROTAC 7’s binding to
the target protein and the E3 ligase-VHL.
In this study, we successfully developed PROTACs from
covalent kinase inhibitors. These covalent inhibitor-based
PROTACs irreversibly bound with target kinases and achieved
excellent degradation potency in live cells. PROTAC 7 effectively
degraded the BTK protein with a DC50 of 136 nM and a Dmax of
88% and also degraded the BLK protein with a DC50 of 220 nM
and a Dmax of 75%. Thus, covalently binding to kinases does
not prevent the formation of effective PROTACs. However,
Fig. 3 The comparison of BTK degradation by covalent and non-covalent
PROTACs. (a) Chemical structures of PROTACs 6b, 7, 9, and 10.
(
b) Western blot results of BTK and GAPDH. K562 cells were treated with our optimization process also clearly indicates that all three
PROTACs for 18 h at the two indicated concentrations. (c) BTK protein components (ligand, linker and E3 ligase) of PROTAC mole-
levels in cells. Numbers were calculated by the BTK/GAPDH ratio with
normalization by the DMSO control as 100. The bars in the graphs show
cules need to be adjusted to obtain good degraders. As covalent
inhibitors with superb binding affinities toward traditionally
the means
Æ
standard deviations from three biological replicates.
druggable and undruggable targets have been developed and
*
P o 0.05 and **P o 0.01 are from unpaired t-tests. (d) IC50 values of
1
0,26
PROTACs 6b, 7, 9, and 10 against BTK kinase.
have achieved success in the clinic,
our results would
strongly suggest adapting them into PROTACs to further extend
the scope of PROTACs.
We acknowledge funding support from the National Natural
Science Foundation of China (81872749), and the Shenzhen Science
and Technology Innovation Commission (JCYJ20160226105227446
and 1210318253).
PROTACs 10 and 6b showed comparable results (Fig. 3b and c). The
IC50 values of these compounds against BTK were also measured
(Fig. 3d). Compared to PROTAC 9, PROTAC 7 is a more potent
inhibitor and a better degrader, and PROTACs 10 and 6b have
comparable potencies in enzymatic assays and degradation abilities
in live cells. The catalytic property is an important and interesting
feature of degraders; some PROTACs with weak binding moieties
can still have potent degradation abilities. PROTACs with a
Conflicts of interest
covalent irreversible binder would likely lose this property. There are no conflicts to declare.
However, the strong binding potencies of covalent PROTACs 7
and 6b appear to compensate well for the loss of turnovers.
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for 18 h to evaluate its ability to degrade the BLK protein.
Indeed, PROTAC 7 potently reduced the BLK protein level with
a DC50 of 220 nM and a Dmax of 75% (Fig. 4b and Fig. S7, ESI†).
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